Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity
ABSTRACT The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to >95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.
Full-textDOI: · Available from: Zachary F Pursell, Aug 12, 2015
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- "in RNase H2 (Lujan et al., 2012). As is displayed in Figure 1B, high-mobility fragments are more abundant when RNase H2 is defective (Figure 1B, left, lane 3, pol2-M644G rnh201D) as compared to when it is not (lane 1, pol2-M644G RNH + ). "
ABSTRACT: RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.Molecular cell 01/2013; 49(5). DOI:10.1016/j.molcel.2012.12.021 · 14.46 Impact Factor
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ABSTRACT: The Saccharomyces cerevisiae EXO1 gene encodes a 5' exonuclease that participates in mismatch repair (MMR) of DNA replication errors. Deleting EXO1 was previously shown to increase mutation rates to a greater extent when combined with a mutator variant (pol3-L612M) of the lagging strand replicase, DNA polymerase δ (Pol δ), than when combined with a mutator variant (pol2-M644G) of the leading strand replicase, DNA polymerase ɛ (Pol ɛ). Here we confirm that result, and extend the approach to examine the effect of deleting EXO1 in a mutator variant (pol1-L868M) of Pol α, the proofreading-deficient and least accurate of the three nuclear replicases that is responsible for initiating Okazaki fragment synthesis. We find that deleting EXO1 increases the mutation rate in the Pol α mutator strain to a significantly greater extent than in the Pol δ or Pol ɛ mutator strains, thereby preferentially reducing the efficiency of MMR of replication errors generated by Pol α. Because these mismatches are closer to the 5' ends of Okazaki fragments than are mismatches made by Pol δ or Pol ɛ, the results not only support the previous suggestion that Exo1 preferentially excises lagging strand replication errors during mismatch repair, they further imply that the 5' ends serve as entry points for 5' excision of replication errors made by Pol α, and possibly as strand discrimination signals for MMR. Nonetheless, mutation rates in the Pol α mutator strain are 5- to 25-fold lower in an exo1Δ strain as compared to an msh2Δ strain completely lacking MMR, indicating that in the absence of Exo1, most replication errors made by Pol α can still be removed in an Msh2-dependent manner by other nucleases and/or by strand displacement.DNA repair 12/2012; 12(2). DOI:10.1016/j.dnarep.2012.11.001 · 3.36 Impact Factor
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ABSTRACT: DNA mismatch repair (MMR) is an evolutionarily conserved DNA repair pathway that plays an essential role in maintaining genomic fidelity and stability. MMR targets errors generated during DNA replication, contributing 100–1,000-fold to the overall fidelity of DNA replication. Inactivating mutations in highly conserved MMR genes greatly increase the spontaneous mutation rate, and loss of MMR predisposes individuals to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. Loss of MMR activity due to epigenetic silencing of MMR genes or somatic mutation is associated with a variety of sporadic tumors. Proteins involved in MMR also participate in DNA damage signaling inducing cell cycle arrest and apoptosis in response to certain DNA alkylating agents and other DNA-damaging agents or base analogs. This review summarizes our current understanding of the MMR pathway and its roles in cancer avoidance.DNA Alterations in Lynch Syndrome, 01/2013: pages 25-45; , ISBN: 978-94-007-6596-2